Oxidative stress induced by reactive oxygen species (ROS) including H202 produced by leukocytes and vascular cells plays a key role in pathogenesis of many disease conditions including atherosclerosis, stroke, hypertension, inflammation, Acute Lung Injury (ALI/ARDS), thrombosis, ischemia-reperfusion injury, organ transplantation, diabetes, angina and myocardial infarction. Therefore, containment of vascular oxidative stress is important to prophylaxis and treatment of these maladies.
Small antioxidants and scavengers can attenuate oxidative stress by terminating lipid peroxidation chain reactions and repairing oxidized molecules in the body, yet they are consumed in these reactions and, therefore, are protective only at very high concentrations. Also, they poorly detoxify directly toxic ROS. Antioxidant inducers, i.e., agents that boost production of natural antioxidants and antioxidant enzymes in the body, also work only at large doses and require pro-longed treatment to develop protective effects. Therefore, while these antioxidant agents may have some utility for alleviating subtle chronic oxidative stress (for example used in form of dietary additions), they have little, if any value for protection against severe acute insults.
In contrast, antioxidant enzymes (e.g., catalase and superoxide dismutase), are not consumed in reactions with ROS, directly detoxify ROS (this preventing the very initiation of oxidative reactions) and are very effective even at very low doses. Therefore, antioxidant enzymes can afford more potent protection, which is critically important for containment of acute and sub-acute severe oxidative stress, such as that occurring in inflammation, stroke, infarction or ALI/ARDS. However, inadequate delivery to endothelial cells lining vascular lumen has hampered their effectiveness for treatment of these and other pathological conditions involving vascular oxidative stress (Muzykantov (2001) J. Control. Rel. 71:1-21).
In order to improve delivery to endothelium, representing both a source of ROS and a critically important, vulnerable target of oxidants (Springer (1990) Scand. J. Immunol. 32:211-216; Varani, et al. (1990) Shock 2:311-319; Heffner & Repine (1989) Am. Rev. Respir. Dis 140: 531-554), diverse means of delivery have been designed (Kozower, et al. (2003) Nat. Biotechnol. 21:392-398; McCord (2002) Methods Enzymol. 349:331-341). For example, targeting of catalase conjugated with antibodies against endothelial cell adhesion molecules ICAM-1 and PECAM-1 boosts vascular antioxidant defense and alleviates oxidative stress in cell cultures (Muzykantov, et al. (1999) Proc. Natl. Acad. Sci. USA 96:2379-2384; Sweitzer, et al. (2003) Free Radic. Biol. Med. 23:1035-1046), perfused organs (Atochina, et al. (1998) Am. J. Physiol. 275:L806-L817), lung transplantation in rats (Kozower, et al. (2003) supra) and lung injury in mice (Christofidou-Solomidou, et al. (2003) Am. J. Physiol. 285:L283-L292). In addition to enhanced delivery of therapeutics, targeting cell adhesion molecules inhibits leukocyte adhesion to the endothelium, thus attenuating their pro-inflammatory functions (DeMeester, et al. (1996) Transplantation 62:1477-1485; Lefer, et al. (1996) Am. J. Physiol. 270:H88-H98; Kumasaka, et al. (1996) J. Clin. Invest. 97:2362-2369)
Studies have revealed that enzymes targeted to endothelial cells (including ICAM-1 and PECAM-1 directed conjugates) enter endothelial cells via a novel internalization mechanism, cell adhesion molecule-mediated endocytosis (Muro et al. (2003) J. Cell. Sci. 116:1599-1609), which provides a pathway for intracellular drug delivery of sub-micron drug-loaded carriers targeted to ICAM-1 or PECAM-1 (Wiewrodt, et al. (2002) Blood 99: 912-922). This enhances detoxification of injurious diffusible intracellular oxidants and minimizes catalase shedding from cell surface (Muro et al. (2003) supra) Using a model polystyrene nanoparticle system with surface-absorbed catalase, it was found that the subsequent intracellular trafficking led to a lysosomal destination and degradation of catalase within 3 hours after delivery, restricting the duration of antioxidant protection (Muro, et al. (2003) Am. J. Physiol. Cell Physiol. 285: C133 9-C134 7). Moreover, other nanoparticle systems are suggested for encapsulation of proteins (see, e.g./U.S. Pat. Nos. 5,543,158 and 6,007,845); however, loading protocols for maintaining functional activity of cargo enzymes are lacking.
Accordingly, there is a need in the art for a delivery system for targeting active therapeutic enzymes and other therapeutic proteins to cells which provides protection of the proteins from subsequent cellular degradation. The present invention meets this need in the art. Furthermore, it establishes a novel class of drug delivery systems based on polymer nanocarriers loaded with encapsulated active enzymes that are not only protected against proteolysis, but capable of carrying out their therapeutic function in the body and inside the target cells without need for drug release from the carrier, due to detoxification of toxic compounds (e.g., ROS) diffusing through the polymer carriers.